Psychostimulant drugs induce long-lasting behavioral changes including dependence and psychosis. The synaptic underlying these behaviors appear to be different than short-term mechanisms that underlie psychostimulant reward. In conjunction with Drs. Fegis Kelly and Robert Edwards, we outline research to elcudiate mechanisms of stimulation- and psychostimulant-induced dopamine synaptic plasticity. We will measure effects of autoreceptor activation on the release frequency of DA vesicles. Using electrochemical techniques to examine release of individual DA vesicles from PC12 cells, we have found that D2 autoreceptor activation rapidly depresses presynaptic DA function by lowering the frequency of stimulation-dependent vesicular release. We will establish whether these effects are due to altered ion conductance or altered vesicle fusion/docking mechanisms and if long-term exposure to D2 agonists promotes sensitization of psychostimulation-or stimulation-dependent release. 2. We will determine the role of VMAT expression in the quantal size of release from DA vesicles. We have recently shown that cocaine and amphetamine, as well as D2 autoreceptor activation, decreases synaptic vesicle-mediated dopamine release by reducing quantal size. Using PC12 lines developed by Dr. Edwards, we will determine if altered phosphorylation of the adrenal gland vesicular monoamine transporter (VMAT1) alters quantal size and if this is differentially modulated by psychostimulants, D2 agonists, and VMAT inhibitors. We will compare results with PC12 lines that express high levels of VMAT1 and lines that express synaptic vesicle VMAT (VMAT2), which has greater affinity for AMPH. 3. We will examine DA release from individual synaptic vesicles of PC12 cells and midbrain neurons. We have performed the first direct measurement of quantal release from midbrain DA neurons; however, this protocol is challenging and not easily amenable to experimental manipulation. The techniques we employ in pC12 cells measure DA release from large PC12 vesicles but not smaller vesicles that more closely resemble conventional synaptic vesicles. Findings by Dr. Edwards suggest that stimulation of VMAT2-transfected PC12 cells release DA from small vesicles; if so, we will observe exocytosis in a preparation amenable to mutation and transgenic manipulation. We will also determine if potential VMAT sorting signal sequences can be used to target VMAT to small vesicles. 4. We will determine effects of neuronal stimulation, autoreceptor activation, and psychostimulants on vesicle sorting and recycling. Dr. Kelly's work suggests that regulation of recycling may control the number and type of secretory vesicles available for release. We will examine recycling in naive and NGF-treated PC12 cells and midbrain DA neurons using differentially labeled components of large dense core granules and synaptic vesicles to determine the roles of acidic recycling intermediates (endosomes). We will extend observations suggesting that depolarization increases intracellular pH gradients, which may contribute tot he rate of membrane recycling. We will determine if psychostimulant and short-term and long-term D2 activation exposure inhibits vesicle recycling.